The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth.Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of −5 °C/min to −20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of −5 °C/min to −20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin.The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034).The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me₂SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me₂SO at −30 °C or with 3% Me₂SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me₂SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me₂SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me₂SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me₂SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.     

Heterologous expression,refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53 °C ± 0.53 °C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34 °C ± 0.25 °C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Optimizing the crystal size and habit of β-sitosterol in suspension

The aim of this work was to survey how processing parameters affect the crystal growth of β-sitosterol in suspension. The process variables studied were the cooling temperature, stirring time and stirring rate during recrystallization. In addition, we investigated the effect a commonly used surfactant, polysorbate 80, has on crystal size distribution and the polymorphic form. This study describes the optimization of the crystallization process, with the object of preparing crystals as small as possible. Particle size distribution and habit were analyzed using optical microscopy, and the crystal structure was analyzed using X-ray diffractometry. The cooling temperature had a remarkable influence on the crystal size. Crystals with a median crystal length of ≈23 μm were achieved with a low cooling temperature (<10°C); however, a fairly large number of crystals over 50 μm appeared. Higher cooling temperatures (>30°C) caused notable crystal growth both in length and width. Rapid (250 rpm), continuous stirring until the suspensions had cooled to room temperature created small, less than 50 μm long (median <20 μm), needle-shaped crystals. The addition of surfactant slightly reduced the size of the initially large crystals. Both hemihydrate and monohydrate crystal forms occurred throughout, regardless of the processing parameters. By using an optimized process, it was possible to obtain a microcrystalline suspension, with a smooth texture.     

Understanding the Influence of State/Phase Transitions on Ice Recrystallization in Atlantic Salmon (Salmo salar) During Frozen Storage

Temperature fluctuations during storage and distribution of frozen foods lead to ice recrystallization and microstructural modifications that can affect food quality. Low temperature transitions may occur in frozen foods due to temperature fluctuations, resulting in less viscous and partially melted food matrices. This study systematically investigated the influence of state/phase transitions and temperature fluctuations on ice recrystallization during the frozen storage of salmon fillets. Using a modulated differential scanning calorimeter, we identified the characteristics glass transition temperature (T _g ′) of −27 °C and the onset temperature for ice crystal melting (T _m ′) of −17 °C in salmon. The temperature of salmon fillets in sealed plastic trays was lowered to −35 °C in a freezer to achieve the glassy state. The temperature (T) of frozen salmon fillets in sealed plastic trays was modulated to achieve a rubbery state (T > T _m ′), a partially freeze-concentrated state (T _g ′ < T < T _m ′) and a glassy state (T < T _g ′). We performed temperature modulation experiments by exposing packaged salmon to room temperature twice a day for 2 to 26 min during 4 weeks of storage. We also analyzed ice crystal morphology using environmental scanning electron microscopy and X-ray computed tomography techniques to observe the pore distribution after sublimation of ice crystals. Melt–refreeze and isomass rounding mechanisms of ice recrystallization were noticed in the frozen salmon subjected to temperature modulations. Results show that ice crystal growth occurred even in the glassy state of frozen salmon during storage, with or without temperature fluctuations. Ice crystal size in frozen salmon was greater in the rubbery state (T > T _m ′) due to the increased mobility of unfrozen water compared to the glassy state. The morphological/geometric parameters of ice crystals in frozen salmon stored for 1 month differed significantly from those in 0-day storage. These findings are important to the frozen food industry because they can help optimize storage and distribution conditions and minimize quality loss of frozen salmon due to recrystallization.     

Ice Recrystallization Behavior of Corn Starch/Sucrose Solutions: Effects of Addition of Corn Starch and Antifreeze Protein III

Knowledge of the behavior of corn starch during frozen storage is necessary to understand more complex systems. In the present study, ice recrystallization in corn starch (0.3% and 3%, w/w)/sucrose (40%, w/w) solution was investigated at −10 °C based on the theory of Ostwald ripening. The addition of corn starch to the sucrose solution increased the ice recrystallization (IR) rate constant. To explore the mechanism causing higher IR rate constant, fluorescence microscopy was used to analyze the distribution of corn starch molecules. Fluorescence micrograph showed corn starch distributed homogenously in the freeze-concentrated phase. Ice crystal size distribution assessment showed that at the same average radius, the addition of corn starch increased the standard deviation of ice crystal size distribution. The findings revealed that the addition of corn starch widened the distribution of ice crystal size, which may be the mechanism causing higher IR rate constant. To inhibit the ice recrystallization process, antifreeze protein type III (AFP III) was added to sucrose solutions with and without corn starch. In the presence of corn starch, 0.01-mg/mL AFP III was enough to significantly reduce the IR rate. Conversely, the samples without corn starch did not show a significant reduction in IR rate constant at the same AFP III concentration. The outcomes revealed that corn starch enhanced the activity of AFP III. The results of this study showed that corn starch increased the IR rate constant, and AFP III supplemented with corn starch was synergistically more efficient in retarding IR rate constant.

     

The formation and distribution of ice within dormant and deacclimated peach flower buds

Abstract A freeze-fixation technique was used to examine the distribution of ice crystals and the pattern of freezing in peach flower buds. In dormant buds, ice crystals formed at localized sites within the bud axis and scales. Ice crystal formation disrupted tissues and mechanical injury from repetitive freezethaw cycles was apparent. There was evidence of ice formation in the floral organs of dormant buds exposed to ?25°C but none observed in buds exposed to either ?5 or ?10°C. The distribution of ice crystals was different in deacclimated buds. In addition to large ice crystals within the subtending bud axis and scales, evidence of large crystals within the developing floral organs was noted. These crystals were most prominent in the lower portions of the developing flower and peduncle, and caused a separation of the epidermal layer from adjacent cells. The distribution of ice crystals within both dormant and deacclimated peach flower buds corroborated the results of previous thermal analysis experiments.     

Quality improvements to mackerel (Scomber japonicus) muscle tissue frozen using a rapid freezer with the weak oscillating magnetic fields

Although freezing is the most popular long-term food preservation method, the formation of ice crystals during the freezing process often degrades the quality of the product. Recently, several reports have argued that oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between −30 °C and −35 °C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p = 0.061), and the increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.     

Visualization of intracellular ice crystals formed in very rapidly frozen cells at −27 °C

Tumor cells of an ascites sarcoma of rat were primarily frozen very rapidly with the original host ascitic fluid at ?27 °C by the spraying method. Frozen specimens were fractured and replicated at about ?100 °C under vacuum by a special spray-sandwich method for freeze-etching, and the morphological appearance of ice crystals formed in and around the frozen cells were observed by electron microscopy.The cells cooled very rapidly at ?27 °C actually froze intracellularly, and intracellular ice crystals ranged from 0.03 to 0.5 μm in grain size due to the initial freezing rate of the specimens. In the cells having granulous intracellular ice crystals less than 0.05 μm in grain size, cytoplasmic organelles seemed to maintain their original structures.We suggested in our previous report that these tumor cells, frozen very rapidly at temperatures above ?30 °C, survived intracellular freezing as long as they remained translucent, and optically no ice crystals appeared within them, as seen in intact unfrozen cells. It may therefore be concluded that the tumor cells frozen very rapidly at temperatures near ?30 °C actually freeze intracellularly and probably maintain their viability as long as the size of individual intracellular ice-crystals is kept smaller than 0.05 μm, although the exact critical size of innocuous intracellular ice crystals is uncertain.     

Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing

The two-step cooling procedure has been used to investigate factors involved in cell injury. Chinese hamster fibroblasts frozen in dimethylsulphoxide (5%, $\text{v}\text{v}$) were studied. Survival was measured using a cell colony assay and simultaneous observations of cellular shrinkage and the localization of intracellular ice were done by an ultrastructural examination of freeze-substituted samples.Correlations were obtained between survival and shrinkage at the holding temperature. However, cells shrunken at ?25 °C for 10 min (the optimal conditions for survival on rapid thawing from ?196 °C) contain intracellular ice nuclei at ?196 °C detectable by recrystallization. These ice nuclei only form below ?80 °C and prevent recovery on slow or interrupted thawing but not on rapid thawing. Cells shrunken at ?35 °C for 10 min (just above the temperature at which intracellular ice forms in the majority of rapidly cooled cells) can tolerate even slow thawing from ?196 °C, suggesting that they contain very few or no ice nuclei even in liquid nitrogen. Damage may correlate with the total amount of ice formed per cell rather than the size of individual crystals, and we suggest that injury occurs during rewarming and is osmotic in nature.     

Ultrastructural appearance of freeze-substituted schistosomula of Schistosoma mansoni frozen by a two-step cooling schedule

Schistosomula of the parasitic helminth Schistosoma mansoni were frozen by two-step cooling, then examined for ultrastructural changes by the freeze-substitution method. Samples were cooled at 1 °C min^?1 to ?20, ?25, ?28, and ?38 °C before being cooled at 10,000 °C min^?1 to ?196 °C. The results showed that progressive partial dehydration of the parasites occurred during slow cooling. Numerous cavities, indicating the presence of intracellular ice crystals, were observed in organisms which did not become shrunken. The sizes of the ice cavities varied between organisms and also within the same cell type in individual organisms indicating that intracellular ice nucleation may occur at any time during the slow cooling step. Some organisms cooled first to ?28 or ?38 °C contained no evidence of ice crystal formation. When correlated with previously reported infectivity studies, the results indicated that successful cryopreservation of schistosomula requires slow cooling to approximately ?30 °C to induce cryodehydration, followed by rapid cooling to ?196 °C to prevent ice nucleation or crystal growth.     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

The effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bone

The effects of cryopreservation on tendon allograft have been reported, but remain unclear, particularly the potential effects on mechanical properties and histological changes by ice crystal formation. There are also few studies about effects of heating for sterilization of tendon. We evaluated the effect of cryopreservation or heating on the mechanical properties and histomorphology of rat bone-patellar tendon-bones (BTBs). BTBs were processed by cryopreservation at −80°C for 3 weeks, or heating at 80°C for 10 min. Tensile testing and histomorphological examination were performed. The cryopreservation of tendons showed less influences on their mechanical properties. When cryopreserved BTBs in frozen state were fixed by freeze-substitution method, many spaces were observed in interfibrillar substances. These results suggest that the collagen fibers of cryopreserved tendons were histomorphologically affected by ice crystals. The heating of tendons completely destroyed the collagen fibers of the tendons and is therefore thought to be inappropriate for the sterilization of BTBs.     

Freezing of tissue-limits for the autoradiographic localization of diffusible substances

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.     

Ice recrystallization in a model system and in frozen muscle tissue

Recrystallization produces modifications on ice crystal sizes during storage and transport of frozen foods, reducing the advantages obtained by quick freezing and inducing physicochemical changes which alter their quality and shorten their shelf life. This process involves the growth of the larger crystals at the expense of the smaller ones, being the interfacial energy, the driving force of the phenomenon. In the present work recrystallization was analyzed using direct microscopic observation of ice crystals in a model solution (0.28 N NaCl) and indirect observation of frozen muscle tissue. The model solution allowed visualization of the interface behavior; from the analysis of the ice crystal frequency distributions, relationships between shape and size of the grains were established. A kinetic model based on the average system curvature was proposed obtaining a satisfactory fitness of the experimental data. Values of the kinetic constants determined at different temperatures allowed the estimation of the process activation energy. In muscle tissues isothermal freeze-substitution was used to observe the holes left by the ice in frozen semitendinous beef muscle stored at -5, -10, -15, and -20 degrees C during long periods of time. A different evolution of the mean ice crystal diameter was observed with respect to the model system. In meat samples, at long storage times, a limit diameter value was reached; this situation has been proved to be independent of temperature and initial size (freezing rate); a theoretical expression based on tissue characteristic parameters was proposed for its evaluation. Activation energy for recrystallization in muscle tissue was also determined, being comparable to values for protein denaturation and quality losses.     

Vitrification induces critical subcellular damages in ram spermatozoa

The aim of the present study was to analyse morphological variations in ovine spermatozoa subjected to different cryopreservation protocols using high resolution imaging techniques. Ejaculates were pooled and diluted in Tris-based extender. Aliquots containing 300 × 10⁶ spz/ml were prepared and evaluated a) after the semen collection and pooling, b) after conventional freezing, c) after vitrification of samples maintained at room temperature (22 °C) prior to vitrification, and d) after vitrification of samples maintained at 5 °C prior to vitrification. Sperm motility, acrosome integrity, DNA fragmentation and morphology were assessed. Subcellular sperm changes were assessed and described by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The maintenance of spermatozoa at 5 °C prior to vitrification and the use of 0.4 M sucrose pointed out lower dimensions of area, length and width than fresh, frozen and sperm maintained at 22 °C prior to vitrification. It was observed that the head width and length are significantly higher (P < 0.0001) in fresh spermatozoa than in the vitrified sperm samples. It could be hypothesized that greater intracellular fluid loss during vitrification could prevent damages in the spermatozoon throughout the reduced ice crystals formation, but mainly by the reduction of extracellular ice crystals due to the physical properties modification obtained when high concentrations of sugars are added. This is the first ultramicroscopic study carried out in ovine vitrified spermatozoa, which confirms the functional sperm alterations previously detected.